PROJECT SUMMARY Lipid homeostasis is essential for cell function and disruptions to lipid homeostasis cause disease. Elevated serum cholesterol is a primary risk factor for heart disease, a leading killer of adults in the United States. Hepatic fatty acid and triglyceride accumulation promote fatty liver disease that progresses to non-alcoholic steatohepatitis, liver cirrhosis and cancer. Type II diabetes mellitus is a major risk factor for developing fatty liver, and alarmingly diabetes is projected to affect one-quarter of the U.S. population by 2050. Understanding regulation of cellular lipid homeostasis will identify therapeutic opportunities for these common diseases. Membrane-bound, basic helix-loop-helix leucine zipper transcription factors called sterol regulatory element-binding proteins (SREBPs) are the central regulators of cellular lipid homeostasis, controlling synthesis and uptake of cholesterol, fatty acids, and triglycerides. In Years 1-15 of this project, we successfully leveraged fission yeast as a simple genetic model for studies of SREBP regulation. We discovered that fungal SREBP is a conserved oxygen-responsive transcription factor required for adaptation to low oxygen and virulence of pathogenic fungi. Our work defined two new paradigms for cellular oxygen sensing: (1) oxygen supply controls sterol synthesis, and (2) oxygen regulates binding of the prolyl hydroxylase Ofd1 to effectors. In a pathway distinct from mammals, activation of fungal SREBP requires ubiquitination by the Golgi Dsc E3 ligase and cleavage by the rhomboid intramembrane protease Rbd2. A second, unidentified protease is required to complete release of the N-terminal SREBP transcription factor from the membrane. The advent of CRISPR/Cas9 technology enables genetic experiments directly in human cells. Accordingly, the current proposal reflects a transition in our studies of the SREBP pathway from yeast to mammalian cells. In Years 16-20, we will (1) complete our description of the yeast SREBP pathway by identifying the second SREBP protease, (2) test whether oxygen also regulates mammalian SREBP, and (3) deploy CRISPR/Cas9 genetics to identify new regulators of SREBP and lipid homeostasis. We propose the following specific aims: AIM 1. TO IDENTIFY THE SECOND FISSION YEAST SREBP PROTEASE. AIM 2. TO TEST WHETHER THE HIF-INSIG2 AXIS REGULATES SREBP IN VITRO AND IN VIVO. AIM 3. TO IDENTIFY NEW REGULATORS OF SREBP2-N USING CRISPR/CAS9 GENETIC SELECTIONS. The impact of the proposed studies is high as we will identify a new enzymatic target for antifungal drug development, define a pathway for hypoxic regulation of mammalian SREBP, and discover novel regulators of LDL receptor expression. Our team has extensive expertise in studies of hypoxia in yeast and mice, and we take innovative approaches in applying our knowledge from yeast to mammalian cells. Given the central role for SREBP in control of lipid homeostasis, our findings will inform both cardiovascular and diabetes research.